At present, DNA typing by analyzing DNA polymorphism is widely employed for purposes of criminal investigations, determination of a blood relation and the like. Nucleotide sequences of DNA of individuals of a same species are similar to each other but are partially different. Such diversity of nucleotide sequences of DNA observed among individuals is called DNA polymorphism, which affects the formation of an individual difference at the genetic level.
One type of DNA polymorphism is a short tandem repeat (STR) or a microsatellite. An STR is a characteristic sequence pattern of a short sequence of about two bases to seven bases repeated several to several dozen times, and it is known that the number of repeating units varies with the individual. Analysis of the combination of numbers of repeating units of STRs at specific loci is called STR analysis.
STR analysis utilizing the property that the combination of numbers of repeating units of STRs varies with the individual is used for DNA typing for the purpose of criminal investigations and the like. FBI (the Federal Bureau of Investigation) and the International Criminal Police Organization have defined ten or more STR loci used for DNA typing as DNA markers and analyze patterns of numbers of repeating units of these STR sequences. Because a difference in the number of STR repeating units arises due to the difference in the allele, a number of repeating units of STR in a DNA marker is referred to as an allele below.
In order to extract a certain amount of DNA of an STR locus used as a DNA marker, PCR (Polymerase Chain Reaction) is performed. PCR is a technique for obtaining a certain amount of a sample of target DNA by specifying certain nucleotide sequences called primer sequences at both ends of the target DNA and repeatedly amplifying only the DNA fragment between the primer sequences.
Electrophoresis is performed to measure the length of the target DNA fragment obtained by PCR. Electrophoresis is a method for separating DNA fragments using the phenomenon that the migration speed in a charged migration path varies with the DNA fragment length (the migration speed is lower as the DNA fragment is longer). As means of electrophoresis, capillary electrophoresis using a capillary as the migration path has been often used recently.
In capillary electrophoresis, a thin tube called a capillary is filled with a migration medium such as a gel, and sample DNA fragments are allowed to migrate through the capillary. The DNA fragment length is determined by measuring the time required for the sample to migrate a certain distance (generally from one end of the capillary to the other). Each sample, namely each DNA fragment, is labeled with a fluorescent dye, and the fluorescence signal from the migrated sample is detected with an optical detector placed at the end of the capillary.
Fluorescent dyes used for labeling different DNA fragments have different spectra, which are not sharp and overlap with each other. Therefore, in the optical detector at the end of the capillary, when DNA fragments labeled with different fluorescent dyes have similar fragment lengths, the spectrum obtained with the optical detector is a linear sum of the spectra of the different fluorescent dyes (referred to as fluorescence spectra below), namely a weighted sum. Accordingly, in order to determine the signal intensities of the respective fluorescent dyes, the linear coefficients of the spectra of the fluorescent dyes constituting the spectrum, that is the weighed values, should be determined from the spectrum obtained with the optical detector. The weighed values correspond to the signal intensities of the respective fluorescent dyes.
In order to determine the signal intensities of the fluorescent dyes, the respective fluorescence spectra should be known in advance. Originally, each fluorescence spectrum is determined exclusively by the fluorescent dye and the migration medium, irrespective of the apparatus, but in an actual apparatus, a fluorescence spectrum imaged on the optical detector shifts due to the positional relation of the capillary and the fluorescence signal detector. Accordingly, when the capillary is replaced, fluorescence spectra must be measured in advance before electrophoresis of the target sample, and calibration for adjusting the positions of the fluorescence spectra (referred to as spectral calibration below) is necessary. In this regard, when electrophoresis is performed simultaneously for two or more samples using a capillary array containing aligned capillaries, it is necessary to measure the fluorescence spectra of each capillary.
Next, an example of spectral calibration of the conventional technology is explained, referring to FIG. 2 and FIG. 3.
FIG. 2 is a figure showing an example of images formed on the optical detector placed at capillary ends. Excitation light of a fluorescent dye obtained by irradiating a capillary end with laser light of a specific wavelength is separated in the wavelength direction with a diffraction grating, and the image is detected with a camera device such as a CCD. The figure shows diffraction grating images obtained by irradiating a capillary array containing eight aligned capillaries ((1) to (8)) with laser light (at the bottom of the figure). In the figure (at the bottom), the vertical axis shows the alignment direction of the capillaries and the horizontal axis shows the wavelength direction, and the figure (on top) is a figure showing the signal intensity along the A-A′ direction of capillary (4) shown at the bottom. The figure (on top) shows the signal intensity distribution, where the vertical axis corresponds to the brightness value indicating the signal intensity and the horizontal axis corresponds to the wavelength. As shown in the figure (on top), the spectrum of a capillary can be obtained as a waveform, where the vertical axis corresponds to the signal intensity in the capillary and the horizontal axis corresponds to the wavelength.
In this regard, FIG. 2 shows an example of spectra measured continuously (as a matter of fact, discretely per pixel) using a diffraction grating, but in practice, the data may be obtained by sampling the spectra above at longer wavelength intervals. For example, as shown in the figure, the brightness values may be obtained only at 20 wavelengths (λ(0) to λ(19)) for each capillary. In addition, averages of the brightness values at wavelengths around λ(0) to λ(19) may be taken.
Moreover, an image may be taken after rapidly changing to a filter which filters only the wavelength region in which the sensitivity to a fluorescent dye is the highest, without using any diffraction grating. Alternatively, camera devices and filters corresponding to fluorescent dyes may be provided to the number of the fluorescent dyes, thereby taking images of the respective fluorescent dyes at the same time. Since such a case corresponds to sampling of spectra at the sample positions corresponding to the respective fluorescent dyes from the spectra shown in the figure, the means of the invention below can be similarly applied.
FIG. 3 is a flowchart of the spectral calibration process. In a step 301, a sample called a matrix standard is subjected to electrophoresis. A matrix standard is a reagent used for performing electrophoresis for the purpose of obtaining fluorescence spectra and obtaining the matrix explained below.
Here, a summary of the matrix standard is given using FIGS. 4A and 4B. FIGS. 4A and 4B are on the supposition that fluorescence spectra of five kinds of fluorescent dye (LIZ, PET, NED, VIC and 6FAM) are obtained. In FIG. 4A, the vertical axis shows the fluorescence intensity and the horizontal axis shows the time, and the five kinds of fluorescent dye have sharp peaks at the times corresponding to the respective fluorescent dyes. Therefore, in order to obtain the fluorescence spectrum of a fluorescent dye, the spectrum at the time at which only the fluorescent dye emits light (in FIG. 4A, t0, t1, t2, t3 and t4) should be obtained. Thus, with respect to the matrix standard, five kinds of DNA fragment with different lengths are labeled with different fluorescent dyes. The information concerning the lengths of the DNA fragments corresponding to the respective fluorescent dyes or the order of the lengths is known.
Next, in a step 302, the intensities of the fluorescent dyes are calculated from the spectra at the respective times obtained by electrophoresis in the step 301. Details of this process will be described below. This process may be conducted at each of the scanning times or after storing spectral data at a certain time interval. An example of waveforms of the fluorescence intensities obtained is shown in FIG. 4A.
In FIG. 4A, waveforms of the signal intensities of the fluorescent dyes (referred to as fluorescence intensities below) are shown in one graph, where the horizontal axis corresponds to the scanning time. There are peaks in the fluorescence intensity waveforms, and the peak times correspond to the DNA fragment lengths. As described above, since DNA fragments with different lengths are labeled with different fluorescent dyes with respect to the matrix standard, one fluorescent dye emits light individually at each peak time (t0, t1, t2, t3 and t4).
Thus, in a step 303, peak times of the waveforms of the fluorescence intensities (signal intensities) of FIG. 4A are detected. This peak detection process will be described below. FIG. 4A shows the results that peak times t0, t1, t2, t3 and t4 have been obtained. As described above, since the order of appearing peak times that correspond to the respective fluorescent dyes is known, the kinds of fluorescent dye corresponding to the respective peak times can be identified. The figure shows the results that LIZ, PET, NED, VIC and 6FAM individually emit light at times t0, t1, t2, t3 and t4, respectively. In other words, the spectra at the times correspond to the fluorescence spectra of the respective fluorescent dyes. Therefore, the spectra at the respective peak times should be obtained (see FIG. 4B).
Then, in a step 304, the matrix described below is calculated using the fluorescence spectra. The matrix is a matrix for obtaining the intensities of the respective fluorescent dyes from the spectral waveforms obtained with a detector. The process of calculating the matrix is called spectral calibration. When there are two or more capillaries, it is necessary to calculate the matrix for each of the capillaries. Moreover, it is necessary to perform spectral calibration every time a capillary is installed or a part is changed.